The goal of this proposal is to analyze the process of myelination at a cellular level. The specific objectives are: 1) to isolate oligodendroglia and maintain them in vitro; 2) to define the biochemical properties of isolated oligodendroglia; 3) to characterize enzymes involved in myelin synthesis or maintenance, specifically galactocerebroside sulfotransferase (e.c. 2.8.2.11) and 2',3'-cyclic nucleotide 3'-phosphohydrolase (e.c. 3.1.4.16). ISOLATION AND CHARACTERIZATION OF OLIGODENDROGLIA - Using bulk isolation methods, oligodendroglia can be isolated from larger mammalian brains such as the lamb, calf or human. When fresh brain is used, oligodendroglia can be maintained in vitro for 3-4 days. Maintained cells will incorporate a variety of amino acids into protein and acetate or galactose into lipids. In particular, galactose is incorporated into galactocerebroside, a lipid markedly enriched in myelin. Two antisera have been raised to isolated cell types, one to rat neurons and another to lamb oligodendroglia. Further characterization of these antisera both in terms of their specificity and the specific cell surface antigens are in progress. Galactocerebroside sulfotransferase, the enzyme involved in the synthesis of sulfatide from galactocerebroside, has been purified 500 fold. There are now only three bands on acrylamide electrophoresis. Attempts to raise antibodies to this enzyme preparation are in progress. 2'-3'-cyclic nucleotide 3' phosphohydrolase has been purified, and now runs as a single band on electrophoresis. This purified enzyme will now be used to raise antibodies. The question to be determined is whether CNP is associated with myelin, and if not, with what membrane fraction. BIBLIOGRAPHIC REFERENCES: Poduslo, S.E. and McKhann, G.M.: The isolation of specific cell types from mammalian brain. In D.B. Tower (Ed.-in-Chief) The Nervous System Volume I: The Basic Neurosciences. New York, Raven Press, 1975. Poduslo, S.E., Tennekoon, G., Price, D., Miller, K., McKhann, G.M.: Fetal metachromatic leukodystrophy: pathology, biochemistry and a study of in vitro enzyme replacement in CNS tissue. J. Neuropath. & Exp. Neurol., Vol. 35, 1976.